Helicity-dependent dissociative tunneling ionization of CF4 in multicycle circularly polarized intense laser fields.

Dissociative tunneling ionization of tetrafluoromethane (CF4) in circularly polarized ultrashort intense laser fields (35 fs, 0.8 × 1014 W cm-2, 1035 nm), CF4 → CF4+ + e- → CF3+ + F + e-, has been studied by three-dimensional electron-ion coincidence momentum imaging. The photoelectron angular distribution in the recoil frame revealed that the dissociative tunneling ionization occurs efficiently when the laser electric field points from F to C. The obtained results are qualitatively consistent with the theoretical predictions by the weak-field asymptotic theory (WFAT) for tunneling ionization from the highest and next-highest occupied molecular orbitals, HOMO (1t1), and HOMO-1 (4t2), respectively. On the other hand, the angular distribution shows clear dependences on the polarization helicity, indicating that the breaking of the C-F bonds is sensitive to the helicity of the multicycle circularly polarized laser fields.

Multielectron-Ion Coincidence Spectroscopy of Xe in Extreme Ultraviolet Laser Fields: Nonlinear Multiple Ionization via Double Core-Hole States.

Ultrafast multiphoton ionization of Xe in strong extreme ultraviolet free-electron laser (FEL) fields (91 eV, 30 fs, 1.6×10^{12} W/cm^{2}) has been investigated by multielectron-ion coincidence spectroscopy. The electron spectra recorded in coincidence with Xe^{4+} show characteristic features associated with two-photon absorption to the 4d^{-2} double core-hole (DCH) states and subsequent Auger decay. It is found that the pathway via the DCH states, which has eluded clear identification in previous studies, makes a large contribution to the multiple ionization, despite the long FEL pulse duration compared with the lifetime of the 4d core-hole states.

Characterization of several amino acid transports and glutamine metabolism in MOLT4 human T4 leukemia cells.

The transport system responsible for glutamine, alanine and glutamate in MOLT4 human T4 leukemia cell line were characterized. Kinetic studies of sodium-dependent glutamine and alanine transport exhibited a single saturable high-affinity carrier with a Michaelis constant of 152 +/- 26 microm and 203 +/- 36 microm and a maximal transport velocity of 960 +/- 165 and 1096 +/- 208 nmol/10(9)cells/min, respectively. Glutamate uptake was less than one-tenth of glutamine and alanine, and linearly increased with glutamate concentration which was mediated by diffusion. 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), known as anion channel blockers, inhibited the sodium-dependent glutamine and alanine transport by 40% at 10 microm. Cellular contents of these amino acids in MOLT4 cells revealed glutamate to be the highest among them despite low glutamate influx. A glutamine metabolism study using whole cells indicated this high conversion rate from glutamine to glutamate, but no conversion to another amino acid. Based on these results, the high glutamate concentration in MOLT4 was speculated to be synthesized from transported glutamine by active glutaminase.

GSH depletion, K-Cl cotransport, and regulatory volume decrease in high-K/high-GSH dog red blood cells.

Thiol reagents activate K-Cl cotransport (K-Cl COT), the Cl-dependent and Na-independent ouabain-resistant K flux, in red blood cells (RBCs) of several species, upon depletion of cellular glutathione (GSH). K-Cl COT is physiologically active in high potassium (HK), high GSH (HG) dog RBCs. In this unique model, we studied whether the same inverse relationship exists between GSH levels and K-Cl COT activity found in other species. The effects of GSH depletion by three different chemical reactions [nitrite (NO(2))-mediated oxidation, diazene dicarboxylic acid bis-N,N-dimethylamide (diamide)-induced dithiol formation, and glutathione S-transferase (GST)-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB)] were tested on K-Cl COT and regulatory volume decrease (RVD). After 85% GSH depletion, all three interventions stimulated K-Cl COT half-maximally with the following order of potency: diamide > NO(2) > CDNB. Repletion of GSH reversed K-Cl COT stimulation by 50%. Cl-dependent RVD accompanied K-Cl COT activation by NO(2) and diamide. K-Cl COT activation at concentration ratios of oxidant/GSH greater than unity was irreversible, suggesting either nitrosothiolation, heterodithiol formation, or GST-mediated dinitrophenylation of protein thiols. The data support the hypothesis that an intact redox system, rather than the absolute GSH levels, protects K-Cl COT activity and cell volume regulation from thiol modification.

Reliable sex identification of dogs by modified PCR/RFLP analysis.

To find definitive RFLP sites for canine sex determination, DNA segments corresponding to parts of the canine ZFX and ZFY genes were amplified by PCR and were directly sequenced. According to the newly defined sequence data, the combination of Haelll and Cfr13I sites was found to be useful for not only identifying the sex of the canine DNA samples but also distinguishing them from the human DNA. Conveniently, these two enzymes worked simultaneously in the same single buffer. The double-digestion of the ZFX/ZFY PCR products with HaeIII and Cfr13I showed banding patterns unique to males and females in Canis familialis. This PCR/RFLP method was confirmed to be applicable to various breeds of dog.

Molecular cloning and expression of aquaporin 1 [correction of aquapolin 1] (AQP1) in dog kidney and erythroblasts.

Complementary DNA of the water channel aquaporin 1 (AQP1) was cloned from dog kidney and erythroblasts. The cDNA amplified from mRNA in dog kidney was 816 bp, the same as that in bovines, but longer by 6 bp than that in humans, mice and rats. The 235-bp fragment cDNA amplified from the mRNA in dog erythroblasts, which was differentiated from peripheral blood, was completely identical to the corresponding sequence of cDNA from the dog kidney. Thus, mature red blood cells from dog may have AQP1 in their cell membranes. The amino acid sequence in dog AQP1 was 91-94% identical to that in the other species mentioned above. Dog AQP1 has six predicted transmembrane domains, two NPA motifs, one mercury-sensitive site and four consensus phosphorylation sites, the same as the other species. However, dog and bovine AQP1 have only one N-glycosylation site, while two glycosylation sites were found in human and rodent AQP1. Xenopus oocytes injected with the mRNA of the dog AQP1 exhibited high water permeability in a hyposmotic medium. Thus, dog AQP1 performs water transport the same as in the other species.

Structural and functional analysis of the LaMDR1 multidrug resistance gene in Leishmania amazonensis.

We determined primary sequences of the LaMDR1 gene in Leishmania amazonensis, a protozoan parasite that causes cutaneous leishmaniasis. The longest open reading frame encodes 1341 amino acids for a protein consisting of two similar halves, each containing six putative transmembrane domains and one ATP-binding domain. The protein has no potential N-glycosylation sites at the extracellular region. The LaMDR1 protein was 91 and 78% identical to the closely related ldmdr1 in L. donovani and lemdr1 in L. enriettii, respectively, revealing less conservation in the C-terminal than in the N-terminal transmembrane domains. Transfection of LaMDR1 conferred a multidrug resistance phenotype to wild-type promastigotes, which exhibited a significant level of resistance to vinblastine, doxorubicin, and actinomycin D, but not to puromycin and colchicine. This drug specificity of LaMDR1 was overlapping with but distinct from that of ldmdr1, suggesting functional diversity of MDR1 proteins among different Leishmania species.

Cutaneous staphylococcal granuloma in a free living zebra (Equus burchelli) in Zambia.

An outgrowth on the left anteriothoracic region behind the elbow joint was seen in a free living Zebra at the time of postmortem examination. The covering skin was ulcerated, nodular, hard with multiple fistula containing yellowish pus. A pure culture of coagulase positive Staphylococcus aureus was isolated from the deep tissue. Histopathology revealed pyogranulomatous dermatitis characterized by eosinophilic amorphous grains including bacterial colonies. This is the first report of cutaneous staphylococcal granuloma in Zebra in Zambia.

K(+)-Cl- cotransport and volume regulation in the light and the dense fraction of high-K+ dog red blood cells.

We examined a chloride (Cl-)-dependent K+ transport (K(+)-Cl- cotransport) and regulatory volume decrease in dog red blood cells with high K+, low Na+, and high glutathione (GSH) content (HK/HG) due to the presence of an Na(+)-K+ pump. The HK/HG cells were separated according to their density, and the age-marker enzyme activities, such as glucose-6-phosphate dehydrogenase and cholinesterase, were determined. Unexpectedly, we found that young cells were heavier (more dense) and smaller in size compared with the old cells, which were lighter (less dense) and larger. The K(+)-Cl- cotransport was nearly 10-fold higher in the most dense cells, representing a 12% fraction of the total population compared with the lightest cohort. Although K(+)-Cl- cotransport in both the dense and the light cells was activated by N-ethylmaleimide, swelling and depletion of cellular divalent cations and the activation of the transport in the dense cells was greater. Both the dense and light cells regulated their volume when they were isosmotically swollen. Therefore, the lower activity of K(+)-Cl- cotransport might not explain the relative large volume in old HK/HG cells. The concentration of GSH and glutamate was higher in the light cells. Thus the higher the GSH and glutamate concentration, the greater the cell volume and the lower the K(+)-Cl- cotransport.

Incidence of dogs possessing red blood cells with high K in Japan and East Asia.

The phenotype of high K (HK) red blood cells, which is an autosomal recessive, was found in dog groups from 10 of 13 breeds or populations in Japan. The incidence of HK was 26 to 38% in the San'in-Shiba, Shinshu-Shiba and Akita breeds, and the gene frequencies of HK ranged from 0.513 to 0.612. The highest incidence (42%) was found in the Jindo breed from Korea, and the gene frequency was 0.652. Two other groups from Korea also possessed this HK variation. However, although HK cells were not found in dogs from Taiwan, Indonesia, Mongolia and Sakhalin, Russia, the HK phenotype is clearly distributed now throughout Japan and Korea.

Heredity of red blood cells with high K and low glutathione (HK/LG) and high K and high glutathione (HK/HG) in a family of Japanese Shiba Dogs.

Forty-two of 81 dogs from a family of Japanese Shiba dogs had red blood cells with a high K and a low Na concentration (HK). Of the HK dogs, 32 were high K and low glutathione (HK/LG) and 10 were high K and high glutathione (HK/HG). These variants were found in both males and females. The phenotype of HK was inherited in a recessive mode as reported earlier. A high incidence of HK/LG dogs was found in this family, and the phenotype was also inherited in a recessive mode. Glutamate (Glu) influx, which defines the cellular glutathione concentration, was lower in HK/LG cells than in HK/HG cells (in some cases extremely low). The fact that the red blood cells of HK/LG dogs have the two varying characteristics of a remaining Na, K-pump and low Glu transport suggests that 2 or more genes may be involved. Since an extremely low Glu influx was also found in normal low K and high Na (LK) red blood cells, the characteristic of low Glu transport also exists in LK cells. The phenotype of low Glu transport may also be inherited in a recessive mode. This family therefore had a very high incidence of homozygous recessive genes which control the phenotypes for the Na, K-pump and low Glu transport.

Na-dependent glutamate transport in high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells.

Na-dependent glutamate (Glu) influxes in the red blood cells of normal low K (LK), high K and high glutathione (HK/HG), and high K and low glutathione (HK/LG) dogs were compared. The ranges of the influxes in LK, HK/HG and HK/LG cells were 1.0-63, 62-174 and 1.3-26 mumol/1 cells per h, respectively. Some LK and HK/LG dogs had red blood cells with extremely low Glu influxes. In cells with extremely low Glu influxes, however, there were clear Na-dependent Glu influxes. In LK, HK/HG and HK/LG cells, the Km of Na-dependent Glu influx with respect to the medium Glu concentration were 17, 20 and 19 mM, respectively, and the half-maximal activation (K1/2) with respect to medium Na concentration was 39, 40 and 42 microM, respectively. By the addition of harmaline, a hallucinogenic alkaloid, the Vmax in LK cells was not affected and the Km was increased, while the Vmax was decreased and the Km increased in HK/HG and HK/LG cells. The Ki value with harmaline by means of Dixon plot in LK cells was 5.2 mM, against 1.8 and 1.9 mM in HK/HG and HK/LG cells, respectively. These results suggest that the difference in the Na-dependent Glu influxes between 2 HK groups was due to the varying quantity, not the quality, of the transporter.

Assay of ornithine carbamoyl transferase activity: modification for application to bovine serum.

An assay procedure for ornithine carbamoyl transferase (OCT), which is known to be a liver-specific enzyme, was modified to be adaptable to bovine serum. The concentration of carbamoyl phosphate and the pH of the reaction reagent solution shifted to 60 mM and 7.2, respectively. These modifications contributed to the augmentation of assay sensitivity. The day-to-day reproducibility was 7.9% (coefficient variation) for serum with high OCT activity and 14.8% for normal bovine serum. The activity was stable for 3-4 months by the storage at -20 degrees C and at least 6 months at -80 degrees C. The OCT activity and other biochemical components including aspartate aminotransferase and gamma glutamyl transpeptidase were measured in sera from 164 dairy or raising cows reared at 7 farms. The normal level of OCT activity in sera from these cows ranged from 9.2 to 25.1 U/l (mean +/- 2SD). By comparison between the farms, the highest mean value of OCT activity was found in a farm where cows were suspected to have some kind of latent liver disease from the data of other biochemical parameters. We conclude that the OCT activity is a very useful diagnostic indicator of liver disease in cattle.

Histometric changes in connective fibers of the lung following paraquat administration in rats.

Pulmonary fibrosis induction by paraquat was studied histometrically using an image analyzer. Rats were given a subcutaneous injection of paraquat (7 or 25 mg/kg) and autopsied 3, 6 or 9 days later. The percentage of the area of connective fibers per unit area, and lung prolyl hydroxylase activity, as a biochemical parameter, were measured. Rats treated with 25 mg/kg of paraquat showed an increase in the percentage of the area of the connective fibers per unit area and also an increase in lung prolyl hydroxylase activity 6 days later. Thus, pulmonary fibrosis was confirmed both morphologically and biochemically. It is concluded that histometric examination using an image analyzer provides a very accurate method for the evaluation of pulmonary fibrosis.

Variant of canine erythrocytes with high potassium content and lack of glutathione accumulation.

Although the cation composition of mature erythrocytes in clinically normal dogs comprises low K concentration and high Na concentration because of lack of a Na/K pump, a dog was found that had erythrocytes with high K and reduced glutathione (GSH) concentrations attributable to the existence of a Na/K pump (HK/HG cells). However, 2 dogs were subsequently found that had erythrocytes with high K concentration and increased Na/K pump activity, but without high GSH accumulation (HK/LG cells). In those HK/LG cells, Na-dependent glutamate and aspartate influxes were about a sixth of the values in HK/HG cells, despite a steep Na gradient, and cellular glutamate concentration was not accumulated in HK/LG cells as it was in HK/HG cells. In the latter cells, glutamate and aspartate accumulated because of high activity of Na-dependent amino acid influxes. Therefore, low concentration of glutamate may be the reason for the low GSH concentration in HK/LG cells. In such cells, however, aspartate and glutamine were accumulated similarly as they were in HK/HG cells. The HK/LG cells also had a defect in amino acid metabolism. This defect was different from that in HK/HG cells. Thus, HK/LG cells might be a new model for study of defects in amino acid transport and amino acid metabolism.

Changes in serum C-reactive protein levels in dogs with various disorders and surgical traumas.

The serum levels of C-reactive protein (CRP) produced as an inflammatory response in dogs with various disorders and surgical traumas were measured by enzyme-linked immunoabsorbent assay and slide reversed passive latex agglutination test (RPLA). The CRP levels were greatly increased 1-2 days after surgery in most of the dogs (n = 29) subjected to surgery. These levels had markedly decreased by the time the sutures were removed. In dogs with various disorders (n = 58), the serum CRP levels at first diagnosis were high in infectious diseases. In dogs from which paired serum samples were examined, the serum CRP usually showed a decrease with improvement in the condition (n = 11) or a terminal increase (n = 4) but, conversely, some showed an increase with improvement in the condition (n = 3).

Erythrocyte K-Cl cotransport: properties and regulation.

Erythrocytes possess a Cl-dependent, Na-independent K transport system cotransporting K and Cl in a 1:1 stoichiometry that is membrane potential independent. This K-Cl cotransporter is stimulated by cell swelling, acidification, Mg depletion, and thiol modification. Cell shrinkage, elevation of cellular divalent ions, thiol alkylation, phosphatase inhibitors, and derivatives of certain loop diuretics and stilbenes are inhibitory. Thus regulation of K-Cl cotransport at the membrane and cytoplasmic levels is highly complex. Basal K-Cl cotransport decreases with cellular maturation, whereas its modes of stimulation and inhibition are variable between species. The physiological inactivation appears to be prevented in low-K animal erythrocytes. In certain human hemoglobinopathies, K-Cl cotransport may be the cause of cellular dehydration and volume decrease. K-Cl cotransport occurs also in nonerythroid cells, such as in epithelial and liver cells of other species. At the threshold of molecular characterization, this comprehensive review places our present understanding of the mechanisms modulating K-Cl cotransport physiologically and pathophysiologically into kinetic and thermodynamic perspectives.